Method and agents for the diagnosis and therapy of chronic inflammatory intestinal disease

ABSTRACT

Methods for diagnosis, early diagnosis, differential diagnosis, assessment of the severity and therapy-accompanying monitoring and prognosis of chronically inflammatory intestinal diseases (Crohn&#39;s disease,  Colitis ulcerosa ), in which the presence and/or the amount of one or more antibodies which bind to ribosomal proteins, in particular P0 and L5, are determined in the serum, plasma, tissue samples and/or stool of a patient who is suffering from a chronically inflammatory intestinal disease or in whom such a disease is suspected. The blocking or removal of such antibodies and/or the influencing of antigen-presenting cells or specifically reactive T-cells which react to ribosomal proteins, such as P0 and L5, by suitable agents can be therapeutically utilized.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Phase application of InternationalApplication No. PCT/EP02/09848, filed Sep. 3, 2002, which designates theUnited States. This application, in its entirety, is incorporated hereinby reference.

The present invention relates to novel methods for diagnosis ofinflammatory intestinal diseases, in particular for the diagnosis of thechronically inflammatory intestinal diseases such as Crohn's disease(CD) and Colitis ulcerosa (CU) and their mixed form which is referred toas Colitis indeterminata, which are based on the first evidence ofdefined antibodies (autoantibodies) in patients suffering from suchdiseases, and therapeutic methods derivable therefrom and agents whichcan be used in such therapeutic methods.

The diseases of the gastrointestinal tract include inflammatoryintestinal diseases in which the intestinal wall and/or mucous membraneof the intestine are inflamed. Enteritis in the narrower sense isdefined as an inflammation of the intestinal wall of the smallintestine, while colitis is defined as inflammation of the mucousmembrane of the large intestine. Inflammatory intestinal diseases havevarious causes, for example may be of an infectious nature or may occuras a result of poisoning, and they may occur as acute or chronicdiseases. Among the inflammatory intestinal diseases, chronicallyinflammatory intestinal diseases are of particular importance for theeconomy and internal medicine owing to their high associated morbidity(disease rate) and mortality (death rate): the diseases known by thenames Crohn's disease (synonym: regional enteritis) and Colitis ulcerosa(synonym: Colitis gravis) and their mixed form Colitis indeterminata.Characteristic symptoms are abdominal pain, diarrhoea and, where thesmall intestine is affected, possibly malabsorption. Intestinalstenoses, internal and external fistulas and abscesses occur incomplications in Crohn's disease, whereas severe blood loss and anincreased risk of the occurrence of carcinomas of the large intestine(about 40% where the disease has existed over 25 years) occur in thecase of Colitis ulcerosa. Clinical characteristics in Crohn's diseaseare the discontinuous attack of the distal small intestine and of thecolon, while a continuous intestinal attack, primarily of the distalsections, is present in the case of Colitis ulcerosa. Histologically,the finding of epithelioid cell granulomas is characteristic of Crohn'sdisease, and the finding of crypt abscesses is characteristic of Colitisulcerosa. 5-10% of patients suffering from the chronically inflammatoryintestinal diseases show characteristics of both clinical pictures(Colitis indeterminata).

On the basis of knowledge to date, the chronically inflammatoryintestinal diseases are multifactorial to a limited extent. The factorsdiscussed include nutritional habits and psychic triggers. A polygeneticpredisposition and a number of external influences, particularly fromintestinal flora, have been confirmed.

On the basis of the chronic course of the disease and variousaccompanying phenomena, said chronically inflammatory intestinaldiseases have for a relatively long time also been discussed from thepoint of view of possible autoimmune reactions. However, autoantibodieswhose occurrence has the sensitivity (characteristic based on patient;for an optimum specificity of 100%, the antibodies are found in allpatients) and specificity (characteristic based on healthy person; foran optimum specificity of 100%, the antibodies are not detectable in anyhealthy persons) required for diagnostic purposes have however not beencharacterized to date. The lack of characterization of antibodiesclosely coupled to the pathological process of the chronicallyinflammatory intestinal diseases has to date also prevented thecharacter of such diseases from being specifically considered aspossible autoimmune diseases in any way in the therapy of said diseases.

The present invention is based on novel discoveries which were made bymeans of a specially designed analytical method of immunological proteinanalysis, which is purely empirical and in which, with the exceptionthat a disease manifesting itself in a specific organ or tissue and tobe investigated might be an autoimmune disease, no additionalhypothetical or theoretical assumptions are made. The analytical methodused is explained in more detail below.

The use of the method led to the result that, in patients in whomCrohn's disease or Colitis ulcerosa have been diagnosed on the basis ofthe clinical findings, antibodies which bind to defined ribosomalproteins which as such have proved to be known, namely the ribosomalproteins P0 and/or L5, are found with high sensitivity and specificity.Their participation in an autoimmune process which takes place inCrohn's disease or Colitis ulcerosa was completely unknown to date.

The experimental findings described herein make it possible for thefirst time to diagnose said chronically inflammatory intestinal diseasesby a corresponding antibody detection in biological samples, inparticular in serum, plasma, tissue samples and/or in the stool, withhigh diagnostic certainty.

The novel discoveries about the nature of the antibodies occurring insaid diseases furthermore makes it possible to develop novel therapeuticapproaches for the treatment of said diseases which are autoimmunediseases in character.

As a first aspect, the present invention therefore provides a noveldiagnostic method, in particular for diagnosis, early diagnosis,differential diagnosis, assessment of the severity andtherapy-accompanying monitoring and prognosis, of chronicallyinflammatory intestinal diseases, in particular Crohn's disease, Colitisulcerosa and their mixed form Colitis indeterminata, which ischaracterized in that the presence and/or the amount of one or moreantibodies which bind to ribosomal proteins, in particular the ribosomalproteins P0 and L5, are determined in the serum, plasma, tissue samplesand/or stool of patients who are suffering from an inflammatoryintestinal disease or in whom such a disease is suspected.

The methods for the determination of said antibodies in a biologicalsample may be any known immunodiagnostic methods which are used fordetecting and for measuring antibodies. Preferably, the antibodies aredetermined with the aid of an immunoassay in which the respectiveribosomal protein (P0, L5) in complete form or in the form of an adduct,phosphorylation product, partial peptide or observed splicing variant,in particular of an intestine-specific splicing variant, is used as anantigen for binding the antibodies sought. For marking the antibodiesspecifically bound from a biological sample, it is then possible to usesome suitable anti-human antibody marked in a manner known per se, or afurther antigen preparation which contains the antigen used for theantibody binding, or a similar specific antigen, in marked form. Ofcourse, it is preferable if the assay used for the antibodydetermination ensures the required sensitivity in the range of theantibody concentrations occurring.

The method of determination can also be carried out using chiptechnology or as a rapid test (point-of-care test). The ribosomalprotein P0 and/or L5 to be used as an antigen may be a human or animalprotein which was enriched or isolated from suitable natural (human oranimal) sources or which has been specifically (recombinantly) producedby a genetic engineering method. Since, for example, the ribosomalprotein P0 is found in identical or immunologically very similar formover many species boundaries, a broad range of possibly suitablestarting materials from which the required antigen preparations can beobtained is available for the provision of suitable assays.

For the provision of novel possible therapies of the chronicallyinflammatory intestinal diseases Crohn's disease and Colitis ulcerosa,it is possible to follow therapeutic approaches which on the one handaim at deactivating (blocking) or removing the antibodies involved inthe autoimmune process or, on the other hand, at influencing thepathological process in a specific manner by producing immunotolerance.For blocking the antibodies, it is possible to use active substances,for example ribosomes, ribosome fractions, ribosomal proteins orfragments or derivatives thereof which bind and deactivate thecirculating antibodies. Such specific binders of said antibodies canalso be used for the preparation of materials for affinity purification,by means of which the pathogenic antibodies can be removedextracorporeally by means of a plasmapheresis.

Alternatively, the proteins P0 and/or L5 can be used in complete form orin the form of an adduct, phosphorylation product, partial peptide,peptide analogue or intestine-specific splicing variant as a therapeuticagent for inducing immunotolerance or inducing blocking of the T-cellreactivity in antigen-presenting cells or T-cells by blocking ormodulation of the antigen presentation.

The results summarized above were obtained by means of the following,generally applicable analytical procedure:

A purely empirical analytical method which is suitable for establishingan involvement of an autoimmune process in basically any diseases, andwhich at the same time provides exact information about the bindingpartners of the autoantibodies occurring in the respective disease, wasdeveloped for identifying antibodies (autoantibodies).

The procedure is as follows:

It is assumed that a disease is based on autoimmune reactions or has anautoimmune component, and it is supposed that the autoantibodiescharacteristic for autoimmune diseases attack tissue structures of thoseorgans or tissues in which the symptoms typical for the respectivediseases are particularly pronounced. In order to test the question asto whether typical autoantibodies are involved in the pathologicalprocess, an immunoglobulin fraction is then first obtained by unspecificaffinity purification from patients who, on the basis of clinicalfindings, are suffering from the disease to be investigated. At the sametime, a corresponding immunoglobulin fraction is obtained from healthypersons. The immunoglobulin fractions obtained from healthy persons andpatients are then bound separately to carrier materials for affinitychromatography, so that two columns characterized by differentimmunoglobulins are obtained for comparative affinity chromatography.

Both columns are then loaded with a tissue extract which can be obtainedfrom healthy tissue but also from a pathological tissue which isattacked in the case of the disease.

If one and the same tissue extract is passed over the two differentaffinity columns, components which undergo a specific binding reactionwith the autoantibodies present on the column are retained from theadded tissue extract when additional autoantibodies typical of thedisease are bound to the affinity column loaded with patientimmunoglobulins.

In the subsequent elution of the columns under conditions in whichantigen antibodies bonds are broken, two different eluates (proteinfractions) are obtained, the eluate from the affinity column with thepatient immunoglobulins optionally containing additional componentswhich were bound to disease-specific autoantibodies present.

If the composition of the eluates is subsequently investigated by 2D gelelectrophoresis, the components of the tissue sample which werepreviously bound by the disease-specific antibodies are detectable asadditional spots of the proteins which were not retained by theautoantibody-free affinity column with immunoglobulins of healthypersons.

The protein spots typical of the disease can then be isolated and can beinvestigated by means of modern methods of protein analysis.

If a tissue fraction from a healthy tissue is used, the additionalproteins found are not necessarily the autoantigens which werecausatively involved in the origin of the autoimmune reaction. If, forexample, the autoimmune reaction is triggered/caused by particularpathological forms of the protein found (e.g. specially processedproteins, organ-specific splicing variants, foreign proteins which, forexample, entered the intestine by an infectious route), this need not bereflected in the binding behaviour of the isolated antibodies againstthe “normal” tissue components used in the test. The exact nature of thepathogenic (triggering) autoantigens is only of secondary importance forthe immunodiagnosis, in which all that is important is the reliableassignment of biomarkers to a pathological process. However, in thelight of the knowledge about disease-specific occurrence of specificautoantibodies, it is of course also possible in principle, with the useof tissue samples of patients, optionally in combination withautoantibodies of the type found, to determine more exact details of thefunctional changes which are actually present in the tissues attackedand which are disease-triggering or occur in the patients in interactionwith the autoantibodies. Such subsequent discoveries can then optionallybe used for further improvement of diagnostic procedures.

In the present case, the inventor's experiments, in which the object wasto obtain knowledge about chronically inflammatory intestinal diseases,were carried out using tissue extracts obtained from the small intestineof (healthy) baboons. The tissue extract of primates (baboons) waschosen owing to its easy availability and as experience has shown thatthere is a very high similarity between the proteins of primates andhumans, which manifests itself as high cross-reactivities with manytherapeutic and diagnostic human reagents.

By working, as described in more detail below, with the tissue extractfrom the small intestine of baboons in combination with affinitycolumns, one of which was loaded with an affinity-purifiedimmunoglobulin fraction of healthy persons while the other had animmunoglobulin fraction of patients suffering from Crohn's diseaseand/or Colitis ulcerosa, it was possible to find, in the eluates fromthe patient affinity columns, two protein spots which occurred only inthe eluates from the affinity columns with the patient immunoglobulinsand which, on the basis of gel electrophoresis, have molecular weightsof about 36 kD and 38 kD, respectively. The protein spots were isolatedfrom the electrophoresis gel and decomposed by trypsin digestion intofragments which were analyzed in a manner known per se by massspectrometry and could be identified by comparison with the data forknown trypsin-treated proteins.

It emerged that both proteins found were known ribosomal proteins, onthe one hand the so-called ribosomal protein P0, which has the knownsequence according to SEQ ID NO:2 (database SWISS PROT Entry RLA0_HUMAN60S acidic ribosomal protein P0; P05388; cf. also N. Fabien et al.,(15), Autoantibodies Directed Against the Ribosomal P Proteins are notonly Directed Against a Common Epitope of the P0, P1 and P2 Proteins, J.Autoimmun. (1999) 13, 103-110; and the literature cited therein, inparticular Wool I. G. et al., Biochimie 73:861-870), and on the otherhand the ribosomal protein L5, which has the known sequence according toSEQ ID NO:1 (database SWISS PROT Entry RL5_HUMAN 60S ribosomal proteinL5; P46777; cf. also J.-M. Frigerio, J.-C. Dagorn, J. L. Iovanna, (26),Cloning, sequencing and expression of the L5, L21, L27a, L28, S5, S9,S10 and S29 human ribosomal protein mRNAs, Biochim. Biophys. Acta 1262(1995) 64-68).

It is true that it was known that both proteins or antibodies directedagainst them play a role in autoimmune diseases, in particular inso-called systemic lupus erythematosus (SLE) (cf. for example J. C.Homberg, M. Rizzetto and Deborah Doniach, (2), Ribosomal AntibodiesDetected by Immunofluorescence in Systemic Lupus Erythematosus and otherCollagenoses, Clin. Ex. Immunol. (1974) 17, 617-628; Thomas L., (1),Labor und Diagnose [Laboratory and Diagnosis], 5^(th) Edition, 1998,824-842; A. Giualis et al., (22), Anti-5s RNA/protein (RNP) antibodylevels correlate with disease activity in a patient with systemic lupuserythematosus (SLE) nephritis, Clin. Exp. Immunol. 1994, 95, 385-389).Antibodies against the protein P0 are clinically determined in thediagnosis of SLE. An assay developed for this purpose is described inU.S. Pat. No. 4,865,970. A similar enzyme linked immunosorbent assay forthe detection and quantitation of antibodies to ribosomal P isfurthermore sold as IMMULISA™ Anti-Ribosomal P Kit, ELISA, IMMCODiagnostics Inc., Buffalo, N.Y. 14228. These assays operate with arelatively short peptide sequence from the carboxy terminus of theprotein P0 and the closely related P1 and P2 as specific binders for theantibodies to be determined. Such an assay can also be used in thediagnosis method according to the invention but is less preferable sinceonly some of the relevant antibodies are found using partial peptides ofsaid type (N. Fabien et al., (22), J. Autoimun. (1999) 13, 103-110).

The fact that autoantibodies which bind to said two ribosomal proteinsP0 and L5 are also found in patient sera in the case of the chronicallyinflammatory intestinal diseases Crohn's disease (CD) and Colitisulcerosa (CU) was completely unknown to date.

P0 is a ribosomal protein which is present in identical form or in theform of ribosomal proteins having a very high homology in numerousorganisms which range from lower organisms, e.g. parasites, to humans.It is known that it occurs in pentamer complexes in the large subunit ofribosomes. Extensive scientific literature exists on the ribosomalprotein P0, to which literature reference is hereby made generally byreference to the examples in the attached list of references, inparticular numbers 1-16. In view of the finding in the presentApplication, it is of particular interest that P0 is overexpressed (7,8) in the case of intestinal carcinomas and carcinomas of the liver,and, depending on the tissues, is also expressed on the surface of cellmembranes (5), which is of interest with regard to the intestinalspecificity of CD and CU. It should furthermore be mentioned thatantibodies against ribosomal P proteins, to which P0 belongs, are alsodiscussed from the point of view of anti-lymphocyte antibodies (3, 4).

There is also extensive scientific literature on the ribosomal proteinL5. In this context, reference is once again made generally to theexamples in the list of references, in particular numbers 18-27. Inconnection with the findings in the present Application, it is ofparticular interest that L5 interacts with numerous proteins, forexample those having an enzyme function (e.g. protein phosphatase-1 (19)or calmodulin kinase-2 (27)), and that L5 furthermore occurs incomplexes with RNA (19-23) and in this complexed form can act as anantigen. It is moreover to be expected that the findings in the presentApplication will give strong new impetus to research.

The discovery and identification of the proteins which bonded only toantibodies detectable in the CD or CU patients are described in moredetails below, reference being made to the attached sequence listings.

The figures show the following:

FIG. 1 shows view of 2D electrophoresis gels which permit a comparisonof the spot patterns of the eluates of an affinity column containingimmunoglobulins from sera of healthy persons (A) with those of anaffinity column containing the immunoglobulins from sera of CD or CUpatients (B). The bordered region shows the position of the firstprotein which can be eluted only from the patient affinity columns;

FIG. 2 shows views of 2D electrophoresis gels which permit a comparisonof the spot patterns of the eluates of an affinity column containingimmunoglobulins from sera of healthy persons (A) with those of anaffinity column containing the immunoglobulins from sera of CD or CUpatients (B). The bordered region shows the position of the secondprotein which can be eluted only from patient affinity columns;

FIG. 3 shows the mass spectrum of the trypsin-digested product isolatedfrom the gel of the 2D electrophoresis according to FIG. 1 (B);

FIG. 4 shows the mass spectrum of the trypsin-digested product isolatedfrom the gel of the 2D gel electrophoresis according to FIG. 2 (B).

1. Preparation of an Extract from the Small Intestine of Baboons

Adult baboons (2-3 years old, 25-35 kg, male and female) were sacrificedby means of intravenous administration of dolethal (10 ml). The smallintestine was removed within 15 min, washed with water, divided intoapprox. 10 g pieces and immediately frozen in liquid nitrogen.

In the further processing, samples of the individual deep-frozen smallintestine tissues were pulverized in a porcelain mortar to give a powderwhile cooling with nitrogen (cf. J. Klose, “Fractionated Extraction ofTotal Tissue Proteins from Mouse and Human for 2-D Electrophoresis”, in:Methods in Molecular Biology, Vol. 112, 2-D Proteome Analysis Protocols,Humana Press Inc., Totowa, N.J.). All further steps were carried out at+4° C. The powder was taken up in 100 ml of buffer (50 mM Hepes, 50 mMNaCl, pH 8) and homogenized by means of a 60 ml potter (from BraunMelsungen) in 5 up-and-down movements at 900 rpm. After subsequentcentrifuging for 1 hour at 100,000 g, the supernatant obtained (tissueextract) was recovered and was used for the further investigations.

2. Preparation of Immunoglobulin Preparations and Affinity Columns

The immunoglobulins were isolated from sera of patients with Crohn'sdisease or Colitis ulcerosa on the one hand and from healthy persons onthe other hand (control sera) by means of an unspecific affinitypurification via protein G agarose. For this purpose, in each case 0.2ml of serum was mixed with 0.2 ml of PBS and then 0.5 ml of protein Gagarose (packed gel) was added. The mixture is incubated for 30 min withgentle shaking and then introduced into a glass column (diameter 0.5 cm)which was closed at the lower end with a fine frit. The column is firstwashed with PBS (5 ml), and bound immunoglobulins are then eluted with20 mM citric acid (pH approx. 2.5) (flow rate approx. 1 ml/min). Theimmunoglobulin eluate is neutralized by adding TRIS-HCl, 1 M, pH 8.0.

The purified immunoglobulin fraction is then oxidized for 20 min withaddition of sodium periodate (final concentration 10 mg/ml). The sodiumperiodate is then removed by desalination by means of an NAP-5 column(Pharmacia) according to the manufacturer's method, PBS being used inthe column and as elution buffer.

The desalinated oxidized immunoglobulin fraction is mixed with Carbolinkmaterial (0.5 ml of packed gel, washed with PBS, from Pierce). Afterincubation for 12 h with gentle shaking, the affinity material obtainedand coated with the respective immunoglobulin fractions is introducedinto a glass column (diameter 0.5 cm) having a fine frit and is washedwith 10 ml of PBS.

3. Working Up the Intestinal Extract by Affinity Purification

In each case 5 ml of a baboon intestinal extract according to 1. areadded continuously and repeatedly at a flow rate of 0.5 ml/min for onehour over in each case an affinity column loaded with an immunoglobulinfraction according to 2. The columns are then washed with 5 ml of PBS.The outflow is continuously monitored for absorption at 280 nm.

The proteins bound to the affinity columns are then eluted with 20 mMcitric acid (pH approx. 2.5). The eluates obtained are then analyzed bymeans of 2-D gel electrophoresis.

4. Proteome Analysis Using the Eluates of the Affinity Columns

In the initial analytical 2D gel electrophoresis, the individual eluateswere separated by means of analytical 2D gel electrophoresis, asdescribed in J. Klose et al., “Two-dimensional electrophoresis ofproteins: An updated protocol and implications for a functional analysisof the genome”, Electrophoresis 1995, 16, 1034-1059. The visualizationof the protein in the 2D electrophoresis gel was effected by means ofsilver staining (cf. J. Heukeshoven et al., “Improved silver stainingprocedure for fast staining in Phast-System Development Unit. I.Staining of Sodium dodecyl gels”, Electrophoresis 1988, 9, 28-32). Thestained gels were dried using a Biorad 583 gel dryer.

For evaluation, the protein spot pattern of an eluate from the affinitycolumn containing the immunoglobulins of healthy persons was comparedwith the protein spot pattern which resulted from a corresponding eluatefrom the affinity column containing immunoglobulins of CD or CUpatients. Protein spots which never occurred in healthy persons but werealways additionally present in the case of patient samples were selectedfor further analytical investigations. FIG. 1 and FIG. 2 show picturesof 2D electrophoresis gels which permit the comparison of the spotpatterns of the eluates of an affinity column containing immunoglobulinsfrom sera of healthy persons (A) with those of an affinity columncontaining the immunoglobulins from sera of CD or CU patients (B). Thebordered regions show the positions of the proteins which can be elutedonly from the patient affinity columns.

Surprisingly, two novel protein spots or protein bands were found in theeluates from the patient column: one spot at a molecular weight of about38 kD and a pI of about 5.5 and a sharp band at the molecular weight ofabout 36 kD, which, however, appeared as an elongated strip-like spot inthe pI direction. The two novel protein spots occurred in all eluatesfrom patient columns but were not visible in the eluates from thecolumns containing immunoglobulins of healthy persons.

The novel specific proteins identified in the protein spot pattern ofthe analytical 2D gel electrophoresis were then prepared by means ofpreparative 2D gel electrophoresis (cf. Klose, loc cit). The stainingwas effected by means of Coomassie Brilliant Blue G250 (cf. V. Neuhoffet al., Electrophoresis 1988, 9, 255-262; and Electrophoresis 1990, 11,101-117).

The protein spots preselected for the further analysis were cut out ofthe gel, using the method described in Courchesne PL et al., In: Methodsin Molecular Biology, Vol. 112:487-513, trypsin-digested and analyzed bymass spectroscopy using mass spectrometric investigations as likewisedescribed and discussed in Courchesne PL, loc cit. The fragment patterns(FIGS. 3 and 4) obtained in the mass spectroscopy (MALDI-MS) of the twonovel spots were then compared with fragment patterns of known proteinsavailable from the databases. Various Internet software programs wereused for this purpose. First, the peptide masses determined by MALDI-MSwere matched with the theoretical peptide masses of all known mammalianproteins. For the protein search (in the Owl.7.2.2001 database using thesearch program Protein Prospector), monoisotopic masses with masstolerances of 100 ppm (P0) or 200 pm (L5) were permitted. The furthersearch was carried out with the NCBinr. database using the searchprogram ProFound in the case of the protein from FIG. 1B (P0), and withthe database NCBI (2001/07/20) using the search program ProFound(species: Mammalia) and a mass tolerance of 200 ppm in the case of theprotein from FIG. 2B.

The two proteins typical of the disease were unambiguously identified asthe ribosomal proteins P0 having the amino acid sequence according toSEQ ID NO:1 and L5 having the amino acid sequence SEQ ID NO:2, saidproteins being known per se.

5. Qualitative Comparison Between Controls and Pathological Samples

The results shown in the following table were obtained by preparingindividual affinity columns according to the above procedure, using ineach case 10 sera of healthy persons (control), 10 sera of CD patientsand 10 sera of CU patients, and treating said columns with theabove-mentioned intestinal extract.

TABLE Sample P0 L5 Control 1 − − Control 2 − − Control 3 − − Control 4 −− Control 5 − − Control 6 − − Control 7 − − Control 8 − − Control 9 − −Contro1 10 − − CD patient 1 + + CD patient 2 + + CD patient 3 + + CDpatient 4 + + CD patient 5 + + CD patient 6 − + CD patient 7 + + CDpatient 8 + − CD patient 9 + + CD patient 10 + + CU patient 1 + + CUpatient 2 − + CU patient 3 + + CU patient 4 + + CU patient 5 − + CUpatient 6 + + CU patient 7 + + CU patient 8 + * CU patient 9 + − CUpatient 10 + +

It is evident from the table that both types of antibodies are to befound simultaneously in most CD and CU patients, but at least one typeof antibody is to be found in all CD or CU patients. On the other hand,such antibodies are not found in any serum of the healthy controlpersons. In the relatively limited group of test persons investigated,the selectivity was thus 100%. When both antibodies were determined andthe evidence of an antibody type appeared to be diagnosticallysignificant, the sensitivity was also 100%.

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1. A method for diagnosis of chronic inflammatory intestinal diseases,wherein the method comprises contacting a biological sample from apatient in whom chronic inflammatory intestinal disease is suspectedwith a molecule that detects an autoantibody directed to a ribosomalprotein selected from P0 and L5, wherein the presence of saidautoantibody indicates chronic inflammatory intestinal disease.
 2. Themethod according to claim 1, wherein the chronic inflammatory intestinaldisease is selected from Crohn's disease and Colitis ulcerosa or theirmixed form designated as Colitis indeterminate.
 3. The method accordingto claim 2, wherein said autoantibody is determined with the aid of animmunoassay in which the respective ribosomal protein in complete formor in the form of an adduct, or a phosphorylation product, is used as anantigen for binding and/or detecting the antibodies to be determined. 4.The method according to claim 1, wherein said autoantibody is determinedwith the aid of an immunoassay in which the respective ribosomal proteinin complete form or in the form of an adduct, or a phosphorylationproduct is used as an antigen for binding and/or detectingautoantibodies.
 5. The method according to claim 4, wherein theribosomal protein is a human or animal protein which is enriched orisolated from natural sources or is recombinantly produced by a geneticengineering method.